The differentiation-specific formation of three isoforms of pectinesterase by the broad bean rust fungus Uromyces viciae-fabae is described. Activity becomes detectable when substomatal vesicles are formed. In crude extracts isoform A contributed 78% of the total pectinesterase activity, and isoforms B and C contributed 20% and 2%, respectively. All three isoforms were found extracellularly in ratios identical to those in extracts. The isoelectric points of the pectinesterase isoforms were 8·4 (A), 5·7 (B), and 4·7 (C) as determined by chromatofocusing. Isoform A had an M r of 33500 and showed a distinct pH optimum at 6·0. The M r of isoform B was 40000; its pH optimum ranged from 5·5 to 7·5. Due to its extremely low activity, isoform C was not further characterized. The possible role of pectinesterases in the infection process of the broad bean rust fungus is discussed.